Impaired release of anti-adhesive ATP from stored RBCs: a novel transfusion lesion Transfusion of red blood cells (RBCs) is a frequently administered and costly therapy. Yet in some patient populations - the critically ill, for example clinical outcomes after transfusion are disappointing, and may worsen as a function of the duration of RBC storage. Biochemical and functional changes in banked RBCs are well established, but few or none are credibly linked to the adverse clinical outcomes in transfusion recipients. We have demonstrated that storage progressively impairs the ability of human RBC to release ATP, and that this impairment promotes the adhesion of RBCs to endothelial cells (ECs) in vitro, and sequestration of RBCs in the lung in vivo, with worsened oxygenation in a novel model of transfusion in mice. This novel proadhesive RBC storage lesion is mediated by the RBC-surface adhesion receptor ICAM-4 (LW) and endothelial v3 integrin, and is prevented by co-infusion of an authentic ATP analog. We hypothesize that the storage- induced deficiency in ATP release from transfused human RBCs contributes to pathophysiology in the host through the locally injurious sequelae of endothelial adhesion of RBCs, compromising O2 uptake and delivery. We will test this hypothesis by accomplishing these Specific Aims: 1) Determine the mechanism by which released ATP inhibits RBC adhesion to endothelial cells. We will extend our novel findings that pharmacological inhibition of panx1 (that blocks RBC ATP release) also promotes RBC adhesion by studying the ability of panx1-deficient RBCs to export ATP and their propensity to adhere to endothelium. We will also determine the mechanism of anti-adhesive effects of RBC-derived ATP by testing the influence of specific purinergic antagonists and the role of ATP-sensitive activation of the relevant adhesion receptors. 2) Determine the effectiveness of strategies to a) replete ATP in stored RBCs and b) promote or mimic ATP-release in vitro and upon transfusion in vivo. We will achieve this aim in endothelial cell adhesion assays in vitro and in the murine human-RBC transfusion model we have recently developed. The influence of post-storage ATP repletion (rejuvenation) of RBCs will be determined in vitro and in vivo, and the influence of co-infusion of ATP analogs will also be determined. These novel studies are expected to accelerate our nascent understanding of how the adhesion of normal RBCs is modulated by the release of ATP, a novel function likely to have broader significance, as in the increased endothelial adhesion of sickle, diabetic and malaria-infected RBCs. The innovative approach of examining the adhesion of both human (and mouse) RBCs in a murine model is expected to accelerate the translation of our findings toward clinical investigation in transfusion medicine. The results of these studies are expected to inform the rational design of strategies to improve the risk-benefit balance for RBC transfusion in critically ill and other anemic patients. PUBLIC HEALTH RELEVANCE: Transfusion of red blood cells (RBCs) for anemia fails to provide a benefit in many patient groups, and may raise the risk in some of death and other problems such as respiratory failure. This project focuses on storage- induced declines in the RBC's ability to release the signaling molecule ATP, and the functional consequences of these progressive changes. In particular, we investigate the link between impaired ATP export by stored RBCs and the harmful tendency for RBCs to stick to blood vessels rather than flowing through smoothly in order to pick up oxygen in the lungs and deliver it to the body. We then investigate whether these deficiencies can be corrected in human RBCs, and in transfused mice.